Hybrid phase lateral flow assay

ABSTRACT

The invention relates to devices for performing single step assays for the determination of the presence or absence of an analyte in a liquid sample, and methods of determining the presence or absence of such analytes using such devices. Devices disclosed comprise a labeled analyte-binding reagent reversibly-immobilized on a non-porous solid material, which solid material is in physical contact with a dry porous carrier bearing an immobilized analyte-binding reagent. Also provided are quantitative assay devices.

This application is a divisional of U.S. Non-Provisional applicationSer. No. 11/088,579, filed Mar. 23, 2005, now allowed, which is acontinuation-in-part of U.S. Non-Provisional application Ser. No.10/854,876, filed May 27, 2004, which claims the priority of U.S.Provisional Application No. 60/555,612, filed Mar. 23, 2004, theentirety of these applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Lateral-flow immunoassays, with their ease of use, speed andreliability, are widely used for self-testing and in the clinicalsetting. Lateral-flow immunoassays are probably the most commonnon-electrical method used in rapid medical diagnostics to detect thepresence of a specific analyte in a liquid sample.

In the general method, a liquid sample suspected of containing theanalyte is applied to a porous carrier. Different porous materials arecommonly used for the porous carrier, and can differ in pore size, flowrate, protein-binding specifications and pre-treatment, etc.Essentially, all of the physical activities (e.g., liquid migration) andchemical reactions take place in the porous carrier, in the followingorder.

First a liquid sample to be tested is introduced to a designated area inthe sampling-end (also referred as the “proximal end” or “wet end”) ofthe porous carrier, for a measured time e.g. S seconds or in a measuredvolume e.g. 2 drops. From this point forward, the liquid sample migrateswithin the porous carrier to the direction of the dry end (also referredas the “distal end”). At the outset of the migration, the liquid sampleis frequently optimized for reaction by means of chemicals e.g. pHagents or buffers, surfactants, and/or blockers impregnated into theporous carrier.

Second, while migrating in the porous carrier, the sample mobilizes alabeled reagent that has been reversibly (temporarily) immobilized inthe porous carrier. The zone where the mobilizable labeled reagent islocated is often referred to as the “labeling zone”, but can be referredto as the “reversible immobilization zone” or “mobilization zone”—theterms are equivalent.

Third, while analyte is reacting with the mobilized labeled reagent, theliquid sample and mobilized labeled reagent migrates further within theporous carrier to the detection zone, (which may also be referred to the“irreversible immobilization zone” or merely the “immobilization zone”)where reagent that binds the same analyte is fixed or immobilized,usually in the form of a line. When analyte is present in the liquidsample, a “sandwich” in the form of the mobilized labeledreagent:analyte:immobilized reagent is formed, and the resultingconcentration of the labeled reagent leads to a visible line appearingin the detection zone, which is indicative of a positive result.

Lastly, remaining sample liquid, together with the rest of the labeledreagent further migrates to a control zone, where a second line appearsindicating that sample has progressed through the detection and controlzones and that the assay has provided a valid test result. The rest ofthe sample and the remaining labeled reagent then migrate to a poroussink. Labeled reagent remaining in the porous carrier (other than in thedetection zone, control zone or sink) makes up any background signal. Insome instances where the migration direction reverses, so called “backflow,” occurs. Furthermore, the porous carrier can be pre-treated withchemicals e.g. surfactants.

Lateral-flow immunoassays can also function on the basis of competitivebinding of the analyte. In these devices, lack of the test linegenerally indicates a positive result.

The most common example of a lateral-flow immunoassay device is apregnancy test. These devices are commonly provided for home use, in aplastic housing with a fibrous or a porous extension, which can be heldto a urine stream to collect urine sample into the housing. The urinesample collected this way then migrates to the porous carrier, whichcontains the labeled reagent and the series of events mentioned abovestarts. The analyte detected in a pregnancy test is Human ChorionicGonadotropin (hCG) and the reagents commonly used are anti-hCGmonoclonal or polyclonal antibodies. The most common labels are gold orlatex particles.

Another known example of a lateral-flow immunoassay device commonlyprovided for home use is an ovulation test, the analyte beingLuteinizing Hormone (LH) and reagents being anti-LH, and the rest of thedevice being similar to a pregnancy test.

A professional format of lateral-flow immunoassay devices commonlyreferred to as cassette tests, have smaller housings and a sampleapplication orifice instead of the fibrous extension. The sample orificeexposes part of the porous carrier where a liquid sample can bedispensed with a pipette, directly to the porous carrier.

A low cost format of lateral-flow immunoassay devices is a dipstick testin the shape of a strip.

A diversity of test designs, against an ever increasing number ofanalytes, especially in the cassette and dipstick format, exists in themarket indicating the wide acceptance of the method.

SUMMARY OF THE INVENTION

The present invention provides a new assay, applicable to all knownanalytes, through a reversible reagent immobilization on a non-poroussolid surface and an irreversible reagent immobilization on/in a porousmedia, wherein non-capillary liquid transport may be achieved on thenon-porous solid surface by means of momentum of sampling and/orgravity. The use of a nonporous surface reduces the opportunities fornon-specific binding of labeled reagent, thereby facilitating its readymobilization by applied liquid sample. The increased binding surfacearea of the porous media enables a higher irreversible immobilizationand therefore signal intensity. The use of a minimal number of devicecomponents decreases production related complications and the cost, andthe use of a non-capillary liquid transport means reduces the residualliquid volume not contributing to the assay chemical reaction andreduces the back-flow.

The present invention also provides improvements in quantitative,dipstick and mid-stream test device designs.

The general reactions between the different analytes, labeled reagents,capture reagents and the control reagents are known to those skilled inart and are not the focus of this invention.

In one aspect, the invention encompasses an analytical test device,which, in the presence of a specific analyte in a liquid sample, iscapable of producing a detectable signal, the device is characterized inthat it comprises a non-porous solid surface and a porous carrier. Thenon-porous solid surface comprises a reversibly immobilized labeledreagent against the analyte in a designated reversible immobilizationzone, also referred to as mobilization zone or labeling zone, and theporous carrier comprises an immobilized reagent against the analyte in adesignated detection zone also referred to as an irreversibleimmobilization zone, or merely as an immobilization zone. The liquidsample taken into the device can initiate the mobilization of thelabeled reagent from the non-porous solid surface, and the labeledreagent-sample mix can move to the detection zone via the porous carrierto produce the signal.

The reversibly immobilized reagent in the reversible immobilization orlabeling zone is loosely attached to the non-porous surface such thatwhen sample is applied the reagent releases from the surface and mixeswithin the sample.

In contrast, the immobilized reagent in the detection, immobilization,or irreversible immobilization zone is designed to remain substantiallyin place throughout the entire duration of the assay.

In another aspect, the liquid sample, taken into the device, moves alongthe non-porous surface through the mobilization zone, by non-capillaryliquid transport which is achieved by means of momentum sampling and/orgravity means until sample reaches the porous carrier, which then drawsthe fluid into and over the porous material by capillary or wickingmeans.

It is a further embodiment of the present invention that either thehousing or the bottom surface of the non-porous solid surface include anattached elevation mechanism or foot which causes the device to rest insuch a manner that after the liquid sample is applied the liquid samplemay flow downhill with the assistance of gravity to thaw the liquidsample through the device.

In another embodiment, the device is capable of producing a separatecontrol signal, either in the detection zone or in an additionaldesignated control zone, to confirm the passage of adequate amount ofthe labeled reagent through the immobilization zone to indicate thecompletion of the assay and/or the ability of the device to produce thesignal in the presence of the analyte.

In other embodiments, the device can have, in addition to the non-porouscarrier bearing a reversibly immobilized labeled reagent and a porouscarrier bearing an immobilized reagent, any of the numerous aspects oflateral flow assay devices known to those of skill in the art. Theseinclude, among others, adaptations that permit the measurement ofmultiple analytes in the same sample.

In another embodiment, the device comprises two non-porous solidsurfaces, positioned parallel to each other, at least one of whichcomprises the labeled reagent in dry state in the mobilization zone. Ina preferred embodiment, the second non-porous surface is sized wider andlonger than the first non-porous surface, such that when the secondnon-porous surface is placed over the first non-porous surface, no edgeof the first non-porous surface extends beyond an edge of the secondnon-porous surface, thus providing improved handling and protection fromundesired wetting in midstream sample application. In such a devicedesign, the sample is taken onto the sample reception zone through anorifice in the enlarged non-porous surface.

In an alternately preferred embodiment, the second nonporoussurface-attached to a device is not sized wider and longer than thefirst non-porous surface but is the same width and is at least as longas the length of the porous surface and may extend in length from theporous surface either partially or entirely covering the non-poroussurface.

In another embodiment, the device comprises a housing with an orifice tointake liquid sample and a window to observe the signal. In a preferredembodiment, at least one element of the housing comprises the labeledreagent in a dry state in the mobilization zone.

It is further preferred that the device in the housing is adapted intovarious handling accessories, designed for different test applications,such as home use, professional laboratory use and patient side use,wherein the device with a minimum housing is contained in differentaccessory devices.

The invention further encompasses an analytical test device in the stripformat, characterized in that the device comprises two parallelnon-porous solid surfaces attached to each other or the continuation ofeach other or overlap with each other, in the sample receiving end ofthe device, forming a sample reservoir.

In one embodiment, the reservoir comprises an orifice on either of thenon-porous solid surfaces, which may provide means for sample dispensingand ventilation to release the liquid sample from the reservoir.

In one embodiment, the device further comprises a sink, distal to and influid contact with the porous carrier, the sink capable of absorbingexcess liquid sample after the sample traverses the test strip.

The invention further encompasses a test device, characterized in thatthe device comprises a housing comprising an evaporation opening for thesink.

The invention further encompasses a test device, characterized in thatthe device comprises a housing assembled into a second housing or intoan accessory device.

The invention further encompasses an analytical test device in the stripformat, characterized in that the device comprises two parallelnon-porous solid surfaces, one of the said surfaces being absent frompart of the sink area thus enabling efficient evaporation.

The invention further encompasses a test strip in a midstream testformat, comprising an enlarged solid surface comprising an orifice, theenlarged surface providing protection from undesired wetting during thenecessary sampling and handling steps.

The invention further encompasses a test device according to precedingaspects, characterized in that either or both sides of the detectionzones are transparent or comprise means to enable the observation of thetest result e.g. a window and all above mentioned solid parts,non-porous or porous, can be attached to each other by known techniquessuch as ultra sonic welding, hot laminating, adhesives or mechanicalmeans.

In another aspect, the invention encompasses an analytical device forthe single step determination of the presence of a specific analyte in aliquid sample comprising a test strip comprising: a first piece ofnon-porous solid surface comprising, in a reversible immobilizationzone, a reversibly immobilized labeled reagent that binds the analyte toform a complex; and a porous carrier comprising, in a detection zone, animmobilized reagent that binds the analyte, wherein the porous carrieris in contact with the non-porous solid surface; wherein liquid sampleapplied to the non-porous solid surface mobilizes the reversiblyimmobilized labeled reagent, whereupon analyte in the liquid sample ispermitted to bind the labeled reagent to form a complex which, togetherwith sample liquid, flows into or onto the porous carrier, wherein theimmobilized reagent binds the complex, thereby generating a detectablesignal indicative of the presence of the analyte.

In another embodiment, the porous carrier further comprises, at a siteseparate from and distal to the site of immobilization of theimmobilized reagent, a second immobilized binding reagent that binds anagent other than the analyte, and wherein binding of the secondimmobilized binding reagent to the agent generates a detectable signalthat confirms passage of liquid sample to the site separate from anddistal to the site of immobilization of the immobilized reagent. Inother embodiments, the device is capable of producing a separate controlsignal, either in the immobilization zone or in an additional designatedcontrol zone, to confirm the passage of adequate amount of the labeledreagent through the immobilization zone and/or the ability of the deviceto produce the signal in the presence of the analyte.

In another embodiment, the device further comprises a porous sampleapplication member. The porous sample application member either overlapsand is in physical contact with the reversibly immobilized reagent onthe non-porous solid material, or overlaps and is in physical contactwith at least a portion of the porous carrier, which portion overlapsand is in contact with the reversibly immobilized reagent on thenon-porous solid material.

In other embodiments, the device can have, in addition to the non-porouscarrier bearing a reversibly immobilized labeled reagent and a porouscarrier bearing an immobilized reagent, any of the numerous aspects oflateral flow assay devices known to those of skill in the art. Theseinclude, without limitation, adaptations that permit the measurement ofmultiple analytes in the same sample.

In another embodiment, the reversible immobilization or mobilizationzone comprises a plurality of labeled reagents. In another embodiment,each of the plurality of labeled reagents binds a different analyte.

In another embodiment, the porous carrier comprises, in the detectionzone, a plurality of immobilized binding reagents. In anotherembodiment, each of the plurality of immobilized binding reagents islocated in a spatially distinct site and each binds a different analyteto generate a signal.

In another embodiment, the device further comprises a plurality of thetest strip assemblies, each member of the plurality bearing reagentsthat identify the presence of one or more different analytes.

In another embodiment, the device comprises a second piece of non-poroussolid material, sized wider and longer than the first non-poroussurface, wherein the second piece of non-porous solid material ispositioned parallel to the first piece, the second piece having anaperture through which sample is introduced, and wherein the secondpiece is positioned such that the aperture is located upstream of thereversible immobilization or mobilization zone and such that the secondpiece provides protection to the first non-porous surface from undesiredwetting during mid-stream sample application.

In another embodiment, the device further comprises a sample reservoir,proximal to the reversible immobilization or mobilization zone andformed from a continuation of the first piece of non-porous solidmaterial or from the juxtaposition of a second piece of non-porous solidmaterial with the first piece of non-porous solid material.

In another embodiment, the device further comprises a housing comprisinga non-porous solid material.

In another embodiment, at least one element of the housing constitutesthe reversible immobilization or mobilization zone, the zone comprisingthe reversibly immobilized labeled reagent in a dry state.

In another embodiment, the flow of liquid sample applied to thenon-porous solid surface, from the site of application to the reversibleimmobilization or mobilization zone and subsequently to the porouscarrier, is provided by the kinetic force of sample application, such asby mid stream sample application, pipetting, or by a swab.

The invention further encompasses an analytical device for the singlestep determination of the presence of a specific analyte in a liquidsample comprising a test strip comprising: a) a first piece ofnon-porous solid material comprising on its surface, in a reversibleimmobilization or mobilization zone, a reversibly immobilized labeledreagent that binds the analyte; b) a porous carrier comprising, in adetection zone, an immobilized reagent that binds the analyte, whereinthe porous carrier is in contact with the non-porous solid surface; andc) a second piece of non-porous solid material, placed over (a) and (b),wherein liquid sample applied to the first non-porous solid materialmobilizes the reversibly immobilized labeled reagent, whereupon analytein the liquid sample is permitted to bind the labeled reagent to form acomplex which, together with sample liquid, flows into the porouscarrier, wherein the immobilized reagent binds the complex, therebygenerating a detectable signal indicative of the presence of theanalyte.

In another embodiment, rather than being reversibly immobilized upon thefirst piece of non-porous solid surface, the labeled reagent isreversibly immobilized in a reversible immobilization or mobilizationzone on the surface of the second piece of non-porous solid materialthat faces the first piece of non-porous material, such that when thetest strip assembly is oriented the first piece of non-porous solidmaterial is on the bottom, and the labeled reagent is reversiblyimmobilized on the underside of the second piece.

It is a further embodiment of the present invention that the detectionzone be placed at the proximal portion of the porous carrier, thus beingcloser to the non-porous mobilization zone.

In another aspect, the invention encompasses a device for the singlestep quantitative measurement of an analyte in a liquid samplecomprising: a) a piece of non-porous solid surface, comprising in areversible immobilization or mobilization zone, a reversibly immobilizedlabeled reagent that binds the analyte to form a complex; and b) a stripof porous material, the strip comprising a binding reagent and being incontact with the non-porous solid surface, wherein the binding reagentis applied to and immobilized on the strip of porous material in adetection area having a longitudinal axis parallel to a flow of a liquidsample, wherein when the liquid sample comprises an analyte-labelconjugate that is bound by the binding reagent, the binding results in asignal, and wherein the distance from the point at which the liquidsample entered the detection zone to the furthest point in the detectionarea at which the signal is detected above background is indicative ofthe amount of analyte present in the sample.

In one embodiment, the detection area is at least 1.5 times as long asit is wide. In another embodiment, the detection area extends over atleast 50% of the length of the porous chromatographic material. Inanother embodiment, the detection area extends over at least 60%, 70%,80%, 90% or over the full length of the porous material.

In another aspect, the invention encompasses a method of detecting ananalyte, the method comprising a) contacting a liquid sample to betested for said analyte with an analytical device as described herein,and b) permitting the liquid sample to traverse the test strip assemblyof the device such that reversibly immobilized, labeled reagent ismobilized and thereafter conveyed to the detection zone of the device,whereat, if analyte is present in the liquid sample, a detectable signalis generated, the signal indicative of the presence of the analyte.Analytical test devices incorporating any or all of the embodimentsdescribed above or elsewhere herein can be employed in the analyticalmethods disclosed herein.

As used herein, the term “test strip” refers to a non-porous solidsurface upon which a reversibly immobilized dried reagent is present,and a porous carrier strip with a detection zone which is in contactwith the non-porous solid material. Test strips can additionally have asink element, and/or a second piece of non-porous solid materialoverlaid upon the assembly of the first non-porous solid material andthe porous carrier. A “test strip” can also include a porous samplereceiving element in contact with the non-porous solid material uponwhich the reversibly immobilized reagent is present. Furthermore, a“test strip” may comprise of one piece of material which throughprocessing or chemical treatment comprises both non-porous and porousphysical properties, or multiple pieces including different materials.

As used herein, the term “non-porous” refers to a surface which does notpermit liquid sample to enter into or to pass through it under normalassay conditions. A non-porous surface does not absorb liquid sampleunder normal assay conditions and is non-chromatographic. A non-poroussurface is preferably inert, impermeable to humidity and low in proteinbinding. For a “non-porous” surface as the term is used herein, anypores present are smaller than the smallest reagent used and/or aresmaller than any component of the sample. By “smaller than” is meantthat the smallest reagent or component of the sample will not passthrough any such pore, in any appreciable amount, under liquid flowconditions applied in the routine or optimal operating conditions forthe device. “Non-porous” materials can be made of any low proteinbinding, non-water absorbing, solid material such as thermoform orthermoplastic polymers (e.g. polystyrene, polyethylene, polycarbonate,polypropylene, fluoropolymer, or polyester, or a combination) or glassor metals or ceramics or composite materials well known to those skilledin art. The non-porous surface can also be made of any material which issurface coated with low protein binding, non-water absorbing materiale.g. Teflon® fluoropolymer resin or Mylar® polyester film coatedcellulose. A porous material, such as certain porous plastics ornitrocellulose, may be made non-porous through pressing or chemicaltreatment such that the surface is rendered smooth and inert, andreagents and liquids are incapable of penetrating the surface of the“non-porous” material.

As used herein, the term “porous” when used in relation to a porouscarrier strip means that the material has pores through which thelargest reagents or sample components for a given immunoassay devicewill pass under liquid flow conditions applied in the routine or optimaloperating conditions for the device. The term “porous” refers also to amaterial with pores which permit or conduct the flow of liquid throughthe body of the material. It is to be understood that “porous” materialscan also permit the flow of liquid across the outer surface of thematerial, as long as they also permit the passage of the liquid throughthe body of the material Porous materials with varying ranges andtolerances of pore size are well known in the art, and include, forexample, nitrocellulose, glass fiber, porous plastics, such aspolyethylene, and various chromatographic papers, among others.

As used herein, the term “open,” when used in reference to a side of atest device assembly, means that an edge is not sealed or adhered toanother, such that air or liquid can pass. Thus, an assembly which is“open on at least three sides” has an unsealed opening at an inlet, anoutlet and at least one side.

As used herein, the term “overlap” means that when an assembly is viewedfrom the side, one material, e.g., a sheet of material, extends over topof another. Overlap can but need not necessarily include completeoverlap, i.e., the top material covers the full length of the bottommaterial.

As used herein, the term “at least a portion of said porous carrier,”when used in reference to overlap by, e.g., a sample receiving member orelement, means that the sample receiving member or element overlaps theporous carrier but does not obscure the detection zone.

As used herein, the term “into,” when used in reference to the passageof a liquid sample and materials dispersed within a liquid sample “into”a porous material, means that the sample penetrates a porous materialsuch that it is carried within the pores of the porous material as theliquid sample flows. The term “into” when used in this manner does notexclude the simultaneous passage of liquid sample over the surface of aporous carrier material “into” which the sample is flowing. In preferredaspects, however, the passage of liquid over the surface of the porouscarrier material is reduced by the presence of a non-porous backinglayer and/or the presence of an additional non-porous material overlaidon the porous carrier material.

As used herein, the term “reversible immobilization” refers to thedeposition of a reagent on a non-porous solid surface in a manner suchthat it is readily solubilized or dispersed by liquid sample as appliedto an assay device as described herein. By readily solubilized ordispersed is meant that at least 80%, and preferably at least 90% oreven 100% of the reversibly immobilized material is solubilized ordispersed into the liquid by the application of liquid sample under theliquid flow conditions applied in the routine or optimal operatingconditions for the device.

As used herein, the term “reversible immobilization zone” refer to thediscrete situs on a non-porous solid material at which the labeledreagent is reversibly immobilized. The reversible immobilization zone isalso referred to herein as the “mobilization zone,” or the “labeling”zone.

By “mobilized” is meant at least the partial dispersal or solubilizationof a labeled reagent from the dry, reversibly immobilized state to themobile state capable of flowing to and through a porous carrier in anassay device as described herein. By “mobile” is meant that the labeledreagent is moving in a lateral direction relative to a surface, forexample, the reagent is moving in or with a flowing liquid.

As used herein, the relative terms “proximal” and “distal” are basedupon the direction of flow—liquid sample flows from the proximal end ofa test strip assembly to the distal end.

As used herein, the term “detection zone” refers to the region orregions on a porous carrier as described herein that compriseimmobilized analyte binding reagent, located in one or more discretelocations. The detection zone is proximal to any control zone or zonesand proximal to the sink. In the quantitative assay embodimentsdescribed herein, the detection zone can extend up to the full length ofthe porous carrier material.

As used herein, the term “in contact” refers to direct contact betweentwo entities, but also includes the use of intervening structuresbetween entities described herein, so long as there is touching orcontact between the elements and/or fluid flow amongst the elements.

As used herein, the term “immobilized,” when used in reference to animmobilized reagent (as opposed to a reversibly immobilized reagent)means that the reagent is not appreciably solubilized or displaced bythe passage of liquid sample under the liquid flow conditions applied inthe routine or optimal operating conditions for the device. By “notappreciably solubilized or displaced” is meant that most of theimmobilized reagent remains attached to the porous carrier during andafter passage of the liquid sample under normal or routine assayconditions.

As used herein, the term “specifically binds” means that an agent bindswith a dissociation constant of 1 μM or less. An agent that“specifically binds” an analyte will preferentially bind that analyte inthe presence of a large number of non-related molecules. With regard tobinding agents, it will commonly be the situation that both a reversiblyimmobilized binding reagent and an immobilized binding reagent willspecifically and essentially exclusively bind the analyte of interest.However, the reversibly immobilized labeled reagent and the immobilizedreagent need not necessarily have specificity for binding only to theanalyte of interest. For example, one can use one reagent that binds aclass of analytes, for example, on the basis of a structural similarityshared by members of the class, and a second reagent that binds theanalyte of interest.

As used herein, the term “spatially distinct” means that the givenregion is sufficiently separated from another region as to be discernedas separate and non-overlapping by eye. Generally, as the term is usedherein with regard to regions on a detection zone, two regions are“spatially distinct” if they are greater than or equal to 0.2 mm apart,e.g., 0.2 mm, 0.3 mm, 0.5 mm, 1.0 mm, etc.

As used herein, the terms “wider” and “longer” mean that a given pieceof material, e.g., a sheet of non-porous material, is at least 1 mmgreater, and preferably more (e.g., 2 mm, 3 mm, etc.), in width andlength than another to which it is being compared.

As used herein, the term “midstream sample application” refers to theapplication of liquid sample by holding the assay device in the flow ofa liquid, e.g., in the flow of urine during urination.

As used herein, the term “sample reservoir” refers to an element of anassay device as described herein that can hold liquid sample at leastsufficient to permit an assay device to provide a test result. A samplereservoir will generally be located at the proximal end of the assaydevice, such that application of liquid sample to the reservoir willresult in application of liquid sample to the non-porous solid surfacenear the reversible immobilization or mobilization zone.

As used herein, a “housing” is a casing surrounding at least thedetection zone of an assay device described herein—the housing will haveat least an aperture for the addition of sample, and for the observationof a test result. An aperture to permit evaporation, e.g., from a sink,can also be present. It is preferred where a housing is used, that thehousing be an active member in the assay device, e.g., by providing as asurface for reversible mobilization and/or the immobilization zone.

As used herein, the term “detected above background” means that a givensignal is greater than the amount of signal one would obtain in an assayrun in the absence of analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not to scale.

Drawing 1 shows one embodiment of the devices described herein. Drawing1A shows the assembled device and Drawing 1B shows the unassembleddevice.

Drawing 2A shows an embodiment of the reservoir in the sampling-end of adevice according to one aspect of the invention. Drawings 2B, 2C, 2D and2E show several alternative design options for the said reservoir.

Drawing 3 shows an embodiment comprising an enlarged non-porous surface.Drawing 3A shows the assembled device, and 3B shows the unassembledunattached device.

Drawing 4A shows the one embodiment of the construction of the housingaccording to one aspect of the invention. Drawing 4B shows the samehousing from the top and Drawing 4C shows the same housing from thebottom. Drawing 4D shows the same housing with no optional componentsand with an alternative placement of the labeled reagent.

Drawing 5 shows examples the accessory devices and housings according toseveral aspects of the invention.

Drawing 6 shows schematic diagrams of several configurations of singlestep assay devices as described herein which, in addition to anon-porous surface having a reversibly immobilized reagent and a porousmaterial having an immobilized reagent, further comprise a porous samplereceiving element. The reversibly immobilized reagent (1) is on thenon-porous surface (2) and the irreversibly immobilized reagent(s) arein the porous material (3). Liquid sample is received by liquid samplereceiving element (4).

Drawing 7A-7D shows schematic diagrams of a quantitative assay deviceembodying one aspect of the invention. That portion of the test striplabeled (a) contains an immobilized detection reagent (the detectionzone), while (b) does not.

Drawing 8 shows a side view of a quantitative assay embodiment asdescribed in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a new assay platform that comprise a hybrid teststrip comprising a non-porous solid surface and a porous carrier. Theuse of a non-porous material for the reversible immobilization oflabeled reagent, which must be readily dispersed or solubilized byliquid sample application if the assay device is to function, avoidsnon-specific interactions that frequently interfere with such dispersalor solubilization. While not limited in usefulness to assays usingparticulate labels, the non-porous character of the material used forreversible immobilization avoids problems caused by particles becomingtrapped within the pores of materials commonly used for reversibleimmobilization and the increased binding surface area of the porousmedia enables a higher irreversible immobilization therefore a highersignal intensity. The use of minimal number of device componentsdecreases production related complications and the cost. The use ofnon-capillary liquid transport means reduces the residual liquid volumenot contributing to the assay chemical reaction and the back-flow. Thedevices described herein are well suited for single step analytedetection, i.e., one need only apply liquid sample to the device inorder to obtain a test result.

The present invention also provides improvements in quantitative,dipstick and mid-stream test device designs.

In one aspect, the reversible immobilization zone is open not only onthe ends into which (inlet) and out of which (outlet) liquid sampleflows, but also on one or more sides and potentially on the top. Thisdesign enhances the relative ease of manufacture. Thus, rather thanbeing a closed system, the immobilization zone is open on at least threesides (inlet, outlet, and one side), preferably four sides (inlet,outlet and both sides), and potentially even five sides (inlet, outlet,both sides and the top, i.e., no second surface on top of the reversibleimmobilization zone. Further, the entire device can be open from thesides, as well as from the inlet and after the detection zone oroptional sink. In addition to ease of manufacture, the openness of, forexample, the distal end of the device, can permit evaporation from thatend which drives the continued flow of liquid. Thus, in one aspect, thedevice is open on three (inlet, outlet and one side) or four (inlet,outlet and both sides) sides.

In one aspect, a single step analytical test device is provided, whichproduces a relevant signal depending upon the presence of a specificanalyte in a liquid sample. The device comprises a non-porous solidsurface and a porous carrier attached to it in fluid communication. Thenon-porous solid surface comprises a reversibly immobilized labeledreagent that specifically binds the analyte, located in a designatedmobilization zone. The porous carrier comprises an immobilized reagentthat specifically binds the analyte, located in a detection zone. Theliquid sample taken into the device can initiate the mobilization of thelabeled reagent, and the labeled reagent+sample mix can move to thedetection zone through the porous carrier to produce the relevantsignal.

In operation, the labeled reagent is not mobilized by the sample liquidpassing through the porous carrier, but rather the labeled reagentdissolves or is dispersed into the liquid sample as the sample passesthe non-porous surface of the mobilization zone, and is then carriedinto or onto the porous carrier by the liquid sample.

As noted, in the single step assay devices described herein, when liquidsample is applied to the devices described herein, that is all that isnecessary to generate an assay result. However, the device can bemodified to permit multi-step assay systems, such as the pre-mix of thesample and labeled conjugate before application. Furthermore, followingthe application of liquid sample to the device, the flow of liquid isessentially continuous. That is, the flow does not substantially stop topermit an incubation before the flow is resumed. The rate of flow canchange, for example, as liquid sample moves from the non-porous solidmaterial to the porous carrier material, but there are no impedimentsdesigned to substantially arrest the flow of the sample.

The lack of such designed impediments enables simplified and low costproduction and provides an assay platform which can deliver resultsfaster. It may also provide less complications for test performance.

In one aspect, the surface of the reversible immobilization zone issubstantially smooth, i.e., the surface is not grooved or etched inorder to, or in a manner that, provides increased surface area orchanges the dynamics of liquid flow. A substantially smooth non-poroussurface tends to release labeled reagent more efficiently than does anirregular surface containing grooves or etchings. With regard to surfacearea, in one aspect, the non-porous surface area occupied by thereversibly immobilized labeled reagent is minimized. While otherarrangements can provide satisfactory results, in this aspect, the“footprint” of the reversibly immobilized labeled reagent is kept assmall as possible. This minimizes the potential for nonspecificinteraction of the labeled reagent with the non-porous surface, and canprovide for a more concentrated mobile label front. It can also beadvantageous to locate the reversibly immobilized reagent on thenon-porous surface very close to the porous carrier material.Preferably, the labeled reagent may be lyophilized or dried under vacuumin the reversible immobilization zone however, air drying of the labeledreagent is most preferred.

The devices and methods involving the novel hybrid phase(non-porous/porous) labeled reagent mobilization/detection assemblydescribed herein can include aspects of other lateral flow assay deviceswell known to those of skill in the art, e.g., mobile and/or immobilecontrol reagents, competitive assay format, etc.

In these and other aspects of the invention, the device can be containedin any housing comprising an orifice or aperture, to which a liquidsample can be introduced to initiate the assay. Such housings are knownand commonly produced from water-impermeable thermoform or thermoplasticmaterials. As discussed elsewhere herein, the housing can play anintegral role in the function of the devices in some aspects.

Another aspect of the invention relates to a reservoir structure fordip-stick tests, wherein the test strip comprises two parallel solidlayers on each wide side of the strip sandwiching the porous carrier,and the said layers, by coming together, form a sample reservoir in thesampling-end of the said strip.

Another aspect of the invention relates to a device designed formid-stream sample loading, wherein the test device comprises an enlargedouter surface with a sampling aperture. The enlarged outer surfaceprotects the dry parts of the device from undesired wetting during themid-stream sampling, and provides means for improved handling. Adisplaceable cap or shroud can optionally be included, to cover thesample application element and prevent unwanted evaporation from thesample application end of the device or to avoid damage to surfaces whenthe device is set down after sample loading.

In another aspect of the invention the device may be modified to performa competition-type specific binding assay. Competition assay techniquesare widely known in the prior art. Specifically the mobilizable reagentcompetes with the reagent (e.g. hCG antigen) in the sample for bindingin the detection zone, therefore if a detectable line is present, thetest is negative for the reagent (e.g. hCG antigen).

In another aspect, the devices of the invention can comprise a housing,wherein at least one element of the housing is also at least part of thenon-porous solid surface comprising the mobilization zone, and thenon-porous solid surface is in liquid contact with the porous carrier.

The housing according to this aspect or others described herein can bemade of a single piece, or multiple pieces. The housing can be in anypractical size and shape and can be made of any non-porous and non-waterabsorbing solid material with low affinity to the labeled reagent,including the known housings commonly produced from water impermeablethermoform or thermoplastic materials. In another aspect of the presentinvention the housing may be made of porous materials such as paper thatis treated to become non-porous in critical areas. A porous housing canbe used advantageously to absorb excess sample or can be used as a sink,in addition to adding structural integrity to the device.

In another aspect, the invention relates to accessory devices and shellhousings wherein, the device in its immediate housing as describedherein, can also be contained in accessory devices or shell housings.

In one aspect, the invention provides an analytical test device, whichis capable of producing a relevant signal dependent upon the presence ofa specific analyte in a liquid sample. Drawing 1 shows the basicconstruction of such a device. Drawing 1A shows the assembled device andDrawing 1B shows the unassembled device. The said device comprises anon-porous solid surface (1) and a porous carrier (2) attached to it.The said non-porous solid surface comprises reversibly immobilizedlabeled reagent specific for the analyte in a designated mobilizationzone (3), and the porous carrier comprises an irreversibly immobilizedreagent against the analyte in a designated immobilization zone (4).Optional control zone (5) also contains an irreversibly immobilizedreagent.

The proximal-end (6), i.e., the wet-end, or sampling-end of the deviceand the distal-end (7), i.e., the dry-end of the device are also shown.

The liquid sample, introduced to the device from the proximal end caninitiate, before contacting the porous carrier (2), the mobilization ofthe reversibly immobilized labeled reagent in the mobilization zone (3)and the label reagent-sample mix can migrate on the solid surface viaporous carrier (2) to the immobilization zone through the incubationzone (9) to produce the signal and further migrate to the optionalcontrol zone (5), producing the control signal, and finally migrate tothe sink zone (10).

The non-porous surface (1), can be made of any low protein binding,non-water absorbing, solid material such as thermoform or thermoplasticpolymers (e.g. polystyrene, polyethylene, polycarbonate, polypropylene,fluoropolymer, or polyester, or a combination) or glass or metals orceramics or composite materials well known to those skilled in art. Thenon-porous surface can also be made of any material which is surfacecoated with low protein binding, non-water absorbing material e.g.Teflon® fluoropolymer resins or Mylar® polyester film coated cellulose.Furthermore, the non-porous surface can be created using any material(porous or otherwise) which is capable of being compressed and orsurface treated), so long as the treated surface properties are suchthat they do not permit liquid sample and/or the assay reagents to enterinto or pass through the surface under normal assay conditions.

The porous carrier (2) can be chosen from a wide variety of commerciallyavailable porous materials, according to its flow rate, pore size,protein binding capacity, blocking requirements, suitability for aspecific analyte and label, thickness and backing layer, all within theknowledge of those skilled in art. The most common types of porouscarrier are nitrocellulose and porous plastics. If necessary to preventnon-specific binding or, for example, to aid in wetting properties, theporous carrier can be pre-treated with blocking agents, e.g. bovineserum albumin, or surfactants, or alternatively can be obtainedpre-treated from commercial sources.

The mobilization zone (3) is essentially coated with the labeled reagentto be mobilized upon wetting. The coating can be done with a number oftechniques known to those skilled in art e.g. spraying or dispensing.

Where desired, the non-porous surface, the porous surface, or both canbe treated with agents (e.g., non-ionic surfactants, etc.) that modifythe flow characteristics of applied liquid sample.

The immobilization zone (4) is essentially coated with the unlabeledcapture reagent against the same specific analyte. The coating can bedone with number of techniques known to those skilled in art e.g.spraying or dispensing. Depending upon the methods used, the coatingwill result in varying degrees of penetration of the immobilized reagentinto the porous carrier. Such penetration is neither required for nordetrimental to the function of the devices described herein. That is,there is generally no negative impact of such penetration, although itcan be desirable, e.g., to provide a higher concentration of immobilizedreagent in a smaller area. The most common method of permanently bindingthe capture reagent to the immobilization zone, is passive absorptione.g. dispensing/spraying, incubating, and drying an aqueous reagentsolution on the porous carrier.

Optionally, the immobilization zone of the porous carrier can becompressed, to slow down the liquid flow and increase theimmuno-concentration, thus increasing the signal strength. Thecompression of the porous carrier in the immobilization zone can beperformed prior to or simultaneously with the application of the capturereagent.

The porous carrier (2) has two basic functions, to regulate themigration of the liquid sample+the labeled reagent mix, and to comprisethe immobilization zone (4) and optionally the control zone (5).

Incubation between the analyte (if present) and the labeled reagenttakes place partly in the mobilization zone (3) and partly through themigration between mobilization and immobilization zones. Therefore,liquid passage time between the mobilization zone (3) and theimmobilization zone (4) through the incubation zone (9) has an effect onthe sensitivity of the device. It is known that by adjusting the lengthof the incubation zone (9) and the flow rate of the porous carrier, thesensitivity of the device can be altered.

The distal portion of the porous carrier after the optional control zone(5) acts as a liquid sink (10).

In a preferred embodiment, the device also comprises a sample receptionzone (8) on the non-porous solid surface (1), comprising sampletreatment reagents, either to be mobilized instantly when wet (e.g.surfactants, salts, pH agents, blockers) or binding agents for moleculesto be blocked from the test system. The treatment agents canadditionally, or in the alternative, be applied to the porous carrier.

In a preferred embodiment, the device also comprises an optional zone(13) and an optional attachment zone (21) on the non-porous carrier,which can be used for production and assembly purposes. The optionalzone (13) can also comprise wetting agents, such as non-ionicsurfactants to promote the liquid migration to within the porouscarrier.

In a preferred embodiment, the device comprises a second non-poroussurface (11) which is attached parallel to the first non-porous surface(1), to enhance the sample application and/or the liquid sampletransport from the optional sample reception zone (8) to the proximalend (14) of the porous carrier and/or to avoid the surface transportover the porous carrier (2) and to avoid premature drying of the liquidsample and/or to improve the robustness of the device.

In a preferred embodiment, the second non-porous surface (11) is shorterthan the first non-porous surface (1) to expose the optional sampleapplication zone (8) and to create guidance to the sample application,in the proximal-end of the device.

In a preferred embodiment, the second non-porous surface (11) or thefirst non-porous surface (1) is shorter than the other one in thedistal-end (7) of the device subject to the invention. Thus exposing apart of the liquid sink (10) to the atmosphere, therefore creating anevaporation surface, enhancing the liquid absorption capacity of thesink (10) and inhibiting back-flow of the liquid sample.

In a preferred embodiment, the inner surface (communicating with theporous carrier) of the second non-porous surface (11) also comprises amobilization zone (16) together with the mobilization zone (3) on thefirst non-porous surface (1) or instead of the zone on the firstsurface. The same is possible between the sample reception zone (15) onthe second non-porous surface (11) and the sample reception zone (8) onfirst non-porous surface (1). It is additionally preferred that when thesecond non-porous surface bears the mobilization zone, the secondnon-porous surface is above the first non-porous surface duringoperation of the device, such that gravity also aids in mobilizing thelabeled reagent into the solution.

In a preferred embodiment, the test device as shown in Drawing 1 isassembled into a housing made of various materials such as thermoformedor molded plastic materials. Such housing materials and designs are wellknown to those skilled in art.

Another aspect of the invention concerns a reservoir structure fordip-stick tests, wherein the test strip comprises two parallel solidlayers on each wide side of the strip sandwiching the porous carrier andthe said layers, that, by coming together, form a sample reservoir inthe sampling-end of the said strip.

As shown in Drawing 2A, both non-porous surfaces (1 and 11) are attachedto the each other or are the continuation of the each other or overlapeach other, at the proximal-end (6) of the device subject to theinvention, therefore creating a reservoir (17) for the sample. Thereservoir (17) can collect the required amount of the liquid sampleinstantly, thus eliminating the need to dip the device for a measuredtime into a liquid sample. The reservoir (17) can be designed in any ofa number of shapes, from bulbous to angular; some alternatives are shownin un-scaled side views in Drawings 2B, 2C, 2D and 2E. The reservoir(17) can also comprise substances similar to that of the optional samplereception zone (8).

In a preferred embodiment, the reservoir (17) comprises an orifice (18)on either of the non-porous solid surfaces, which can provide means forsample dispensing and ventilation to release the liquid sample from thereservoir (17).

Another aspect concerns a device optimized for midstream samplecollection, wherein the test device comprises an enlarged surface with asampling orifice. The said enlarged surface protects the dry parts ofthe device from undesired wetting during the mid-stream sampling andproviding means for improved handling.

As shown in Drawing 3, as an assembled device in Drawing 3A and as anunassembled device in 3B, the second non-porous surface is sized widerand longer (19) than the first non-porous surface (1), thus providingimproved handling and protection from undesired wetting in mid-streamsample application. In such a device design, the sample is taken to thesample reception zone (8) through the orifice (20) on the enlargednon-porous surface (19).

In a preferred embodiment, the enlarged second non-porous surface (19)can be attached to the optional attachment zone (21) in the distal endof the first non-porous surface and to the porous carrier (2) on theupper surface of the porous carrier. An optional spacer (22) can be usedto maintain an even distance between the surfaces through the samplereception zone (8).

In a preferred embodiment, a third layer can be attached to a devicewith normal sized non-porous surfaces (Drawing 1 and 2) in the samemanner.

Another aspect, concerns a housing, wherein at least one element of thehousing is also part of the non-porous solid surface comprising themobilization zone. In this aspect, as in the first aspect, the saidsolid surface is in liquid contact with the porous carrier.

The housing subject to invention can be made of a single piece ormultiple pieces. The housing can be in any practical size and shape andcan be made of any non-porous and non-water absorbing solid materialwith low affinity to the labeled reagent including the known housingscommonly produced from water impermeable thermoform or thermoplasticmaterials. However, it is further possible to have a porous housing madenon-porous in critical areas such that the porous nature of the housingcan advantageously be used to absorb the excess applied sample or can beused as a sink.

The device with housing shown in see-through side view in Drawing 4A iscomposed of a top (23) and a bottom (24) housing parts and a porouscarrier (2). The inner surfaces (25 and 26) of both or either partscomprise the mobilization zone (3). The liquid sample is taken into thedevice through the intake orifice (27). The excess of liquid taken intothe device is drained out through the optional drain orifice (28). Theinner surfaces (25 and 26) between the intake orifice (27) and themobilization zone (3) are the sample reception zone. There can be anoptional evaporation grill (29) on the distal bottom end of the device,to assist the sink function. The porous media can, optionally be foldedover itself (30), to create a shorter device design, without decreasingthe sink capacity.

Drawing 413 shows the same device from the top and Drawing 4C shows thesame device from the bottom. Drawing 4D shows in see-through side view,the same housing without optional drain orifice (28), and other optionalcomponents, e.g. folded porous carrier (30) or evaporation grill (29).Also shown in Drawing 4D is an alternative placement of the mobilizationzone (3).

Another aspect concerns accessory devices and shell housings wherein thedevice in immediate housing or in other words, a primary housing,subject to the invention can also be contained in accessory devices orshell housings, in other words, secondary housings.

Drawing 5 shows examples of such an adaptation, wherein the device withan immediate housing (Drawing 4) is contained in a molded plastic shellfor home use (32) or on/in a dip stick extender (34) or in a bench topdevice (33) or used as it is (31). Adaptation can be achieved by asimple receptacle slot (35) in the accessory device as an example shown(35) in cut-away view from inside of any of these accessory devices.

For all aforementioned devices, it will be apparent to those skilled inart that, to enable signal observation, either or both sides of thesignal producing zones should be transparent or should comprise means toenable the observation of the test result e.g. a window, and all abovementioned solid parts, non-porous or porous can be attached to eachother by known techniques such as ultrasonic welding, hot laminating,adhesives or mechanical means.

Examples 1. Non-Porous Solid Surface Material:

The non-porous solid surface can be chosen from wide variety ofmaterials e.g. polystyrene, polyethylene, polycarbonate, polypropylene,or polyester, or a combination, available from numerous commercialsources e.g. General Electric, USA in sheets in a variety of forms andthicknesses.

2. Porous Carrier:

The porous carrier can be chosen from a wide variety of chromatographicmaterials e.g. cellulose, nitrocellulose or porous plastic, availablefrom numerous commercial sources e.g. Whatman, UK; Schleicher & Schuell,Germany and Porex, USA. Non-limiting examples include hydrophilic porouspolyethylene membrane, for example with pore size between 2 and 8micrometer, or lateral flow test nitrocellulose membrane.

3. Label Material:

The label can be chosen from wide variety of materials commonly known tothose skilled in art, e.g. metals, dyes, polymers, enzymes andfluorophores. Use of colloidal gold and latex polymer particles are themost common.

Colloidal gold can be obtained from commercial sources e.g: BBI, UK. Thegold particles can also be produced, for example, according to U.S. Pat.No. 4,313,734. As an example, colloidal gold particles of approximately40 nm in diameter can be used.

Polymer particles can be obtained from numerous commercial sourcescommonly known to those skilled in art e.g. Bangs Laboratories, USA.

4. Binding Reagents:

The binding reagents may be antigens, antibodies or other bindingpartner arrangements (Avidin-Biotin). Antibodies may be useful asbinding reagents, and can be monoclonal or polyclonal obtained fromnumerous commercial sources, commonly known to those skilled in art.Alternatively, antibodies can be raised to a desired antigen accordingto methods well known in the art.

Labeling and capture antibodies can be obtained, for example from Medix,Finland (clone codes: 5006 and 6601) for hCG in case of a pregnancytest.

A control antibody against the labeling antibody, for example, can beobtained from Dako, Denmark (code: Z 0109).

It should be understood that while antibodies (or, for example,antigen-binding fragments of antibodies) are preferred binding reagents,the binding reagents can be selected from any reagent that binds orspecifically binds the analyte of interest. Thus, agents such as nucleicacids, including, but not limited to aptamers, as well as specificbinding partners, e.g., a polypeptide with a complementary bindingdomain for a target protein, such as a leucine zipper or SH2 domain, arecandidates for use in devices and methods as described.

5. Conjugation of Antibody to a Label:

Conjugation of antibody to a label is a common practice to those skilledin art described, e.g. in U.S. Pat. No. 4,313,734 or U.S. Pat. No.5,571,726. Gold conjugates can be prepared by the adsorption of antibodyto the gold surface within minutes through charge attraction,hydrophobic attraction, or sulfur binding.

Polymer conjugates can be prepared by adsorption or covalent binding ofantibodies to the particles according to known methods e.g. BangsLaboratories TechNotes 204 (adsorption to microspheres), 205 (covalentcoupling), and-303 (lateral flow tests).

Alternatively pre-conjugated antibodies can be obtained from numerouscommercial sources e.g. Arista, USA, and British Biocell, UK.

6. Reversible Immobilization of Labeled Antibody to the Non-Porous SolidSurface:

Reversible immobilization solution was obtained from British Biocell,UK, and contained 40 nm colloidal gold coated with anti hCG antibodiesat optical density 10. If desired, solubilizing agents such as trehalose(Sigma, T-5251) can be added to the reversible immobilization solution.

1 microliter of this solution was dispensed per mm width of the solidsurface, to the designated mobilization zone (3) on non-porous surface.Drying was done in room temperature under 20% relative humidity, overnight.

Stabilizing systems or buffers for mobilizable dried reagents are known(see, e.g., Brooks et al., 1986) and are also commercially available(e.g., from Surmodics, U.S.A.). The use of such buffer systems canenhance the stability of labeled reagents in the dry state and canimprove the mobilization characteristics of such reagents.

The non-porous surface material used was a transparent polycarbonatesheet of 200 micrometer thickness. The sheet was cut into strips of 5 mmwidth in varying lengths.

7. Immobilization of Unlabeled Antibodies onto the Porous Carrier:

Monoclonal antibody solution against the alpha-subunit of hCG at 5 mg/mlwas dispensed at 0.2 microliter/per mm width of the porous carrier ontothe designated immobilization zone (4) in surfactant-treated porouscarrier and dried at room temperature under 20% relative humidity,overnight.

Anti-mouse control antibody was also immobilized using the sameprotocol.

The porous member can be treated with surfactants or other wetting,blocking or optimizing agents if desired.

8. Assembly:

The transparent non-porous solid surface was cut in the shape of arectangular strip of 55 mm×5 mm. From the proximal end, at the 10thmillimeter to 55th mm the porous carrier (5×45 mm) was attached usingwater-resistant double-sided transparent adhesive tape. A secondnon-porous solid surface was cut into a strip of 5×60 mm and attached tothe porous carrier, parallel to the first non-porous surface, usingadhesive tape.

Thus, the structures shown in Drawing 1 were achieved.

In another assembly format, the transparent non-porous solid surface wasin the shape of a rectangular strip of 121 mm to 5 mm. From the distalend to the 45th millimeter the porous carrier similar to the previousassembly was attached using water-resistant double-sided adhesive tape.Between the 60th and 61st millimeters the solid surface was bent twiceat 90 degrees and was attached to the porous carrier usingwater-resistant double-sided adhesive tape.

Thus, the structure similar to what is shown in Drawing 2A was achieved.

In another assembly format, the transparent non-porous solid surface wascut in the shape of a rectangular strip of 70 mm by 5 mm. From theproximal end, 25th millimeter to 70th millimeter, the coated porouscarrier was attached using water-resistant double-sided adhesive tape.

A second transparent non-porous solid surface, made of the samematerial, was cut in a rectangular shape of 120 mm by 50 mm. From theproximal end, a hole of 3 mm diameter was punched between the 12th and15th millimeters on the longitudinal axis. Using double-sided tape overthe porous carrier and on the proximal 5 mm of the aforementioned stripstructure, the strip structure was attached to the under side of therectangular sheet, in such a way that the sample receiving zone of thestrip was exposed through the hole.

Thus, the structure shown in Drawing 3 was achieved.

9. Assay for hCG:

To test the function and sensitivity of the assay kits as disclosedherein, a lyophilized hCG standard (calibrated against World HealthOrganization International Standard 75/537) was diluted in PBS. 20 to 80microliters of freshly-prepared hCG dilutions (20IU hCG/l, 100 IU/l,20000 IU/l, and 20000 IU/l) were pipetted to the sample reception zoneof the device for each of 96 identical devices (total of 384 devicesused for hCG dilutions). As a control, 80 microliters of PBS werepipetted to another 20 devices.

All devices with PBS and 20 IU hCG/l gave no visible positive signal.

All devices with 100 IU hCG/l or higher gave visible positive signal.

It is noted that the devices described herein have sensitivity effectivefor diagnosis of pregnancy at its earliest stages.

10. Devices Including a Porous Sample Receiving Element.

In one aspect, devices embodying the non-porous/porous hybrid phaseassembly described herein can include a porous sample-receiving elementas shown in Drawing 6. Referring to the thawing, in one embodiment, theassembly includes a non-porous surface (2) upon which labeled reagent(1) is reversibly immobilized, adjacent to a porous carrier material (3)that can be attached to the non-porous surface (2). A poroussample-receiving material (4), e.g., filter paper, such as Whatman paperor a porous plastic material or other materials commonly used as sampleapplication pad materials for lateral flow assay devices, is placed suchthat it overlaps or abuts with the non-porous surface. Several differentarrangements with regard to the placement of the sample-receivingelement and the reversibly immobilized reagent are shown in panels A-Dof Drawing 6. As shown in Drawing 6A, the porous sample-receivingmaterial can overlap the reversibly immobilized labeled reagent (1).Alternatively, as shown in Drawing 6B, the porous sample-receivingelement (4) can overlap both the reversibly immobilized labeled reagent(1) and the porous carrier material (3). In another alternative, shownin Drawing 6C, labeled reagent (1) is reversibly immobilized on thenon-porous surface (2), the porous carrier material (3) overlaps thereversibly immobilized labeled reagent (1), and the poroussample-receiving element (4) overlaps the porous carrier material (3).In yet another alternative, depicted in Drawing 6D, the labeled reagent(1) is reversibly immobilized on the non-porous surface (2) adjacent to,and preferably, abutting or in contact with the porous sample receivingelement (4) on one (upstream) side and the porous carrier material (3)on the other (downstream) side.

11. Quantitative Assay Format:

In another aspect, a quantitative lateral flow assay is provided. Inthis aspect, rather than immobilizing a capture reagent in a relativelynarrow discrete line perpendicular to the flow of the liquid sample, thenon-reversibly immobilized reagent is applied in a larger detectionarea, longitudinal and parallel to the direction of liquid flow.Examples are depicted in Drawing 7, in which the detection zone,comprising the non-reversibly immobilized capture reagent is appliedessentially to the length of a porous material, which is one option (itis important to note that there is no requirement, according to thisaspect, that the detection area span the entire length of the porousmaterial).

The detection area is coated with non-labeled reagents as used in thedetection zones of qualitative assay devices as described herein, butpreferably in lesser concentration and over a larger area, i.e., not ina narrow line. The labeled reagents useful in this assay format are thesame as would be used in the qualitative assay formats.

The detection area can cover the entire width of the material, as shownin Drawing 7B, or, alternatively, just a strip on the material, as shownin Drawing 7A, (a). Where the arrangement of Drawing 7A is desired, itcan be achieved, for example, by making the remainder of the membrane(b) hydrophobic or non-porous, which thereby directs the flow of liquidsample into a narrow strip. This arrangement can increase the flow, andthus, the immuno-concentration on the longitudinal (strip-like)detection area.

The detection arrangement described above and depicted in Drawings 7A-7Dcan be incorporated into an assay device comprising a reversiblyimmobilized, labeled reagent either as described herein (reversiblyimmobilized on a non-porous surface) or as is known in the art (forexample, reversible immobilization on the chromatographic material,e.g., through use of a glazing material, e.g., a sugar, protein orpolymer, or reversible immobilization in a material such as a glassfiber filter or porous plastic which releases particulate label morereadily than the chromatographic material used in the detection area,among others). While particulate labels are preferred, devices accordingto this aspect, like the others described herein, are not limited to theuse of particulate labels. Alternative labels can include, for example,dyes, such as fluorescent dyes, enzymes, isotopes, etc.

In use, following the application of liquid sample which mobilizesreversibly immobilized labeled reagent (e.g., particle labeled antibodyagainst the analyte), the labeled reagent, complexed with analyte, ifpresent, migrates onto the detection area. Labeled reagent-analyteconjugate will be trapped by the immobilized detection reagent (e.g., asecond antibody that binds the analyte) as the conjugate migrates overor through the detection zone. The conjugate will tend to bind theimmobilized detection reagent in the proximal region of the detectionarea (that region first coming in contact with the liquid samplecarrying the conjugate) first, giving a detectable signal. Quantitationis achieved based on how far distal to that first proximal region thesignal is detected above background. That is, as the immobilized reagentin the proximal detection area is saturated for binding, unboundconjugate will continue along the detection zone until free immobilizeddetection reagent is encountered. The more analyte present, the furtheralong the detection area (distally) the signal will be detected. This isdepicted schematically, for example, in Drawing 8. As an example,gold-conjugated anti-hCG, bound to hCG present in the sample, is trappedby proximal capture antibodies first, and how far distally the colorsignal goes will depend purely on the concentration of the analyte inthe sample.

To aid in interpretation, the detection area for the quantitativeembodiments can be, for example, graduated, as shown in Drawing 7C, orit can contain a cut-off point as shown in Drawing 7D. Alternatively, orin addition, the detection area can be arranged as successive parallellines of unlabeled binding reagent, each separated by a small gap, suchthat the detection area is graduated, permitting quantitation of analytebased upon the number of lines showing bound label. Assays in thequantitative format can, of course, also contain a control reagent zoneas used in other lateral flow assay devices.

Additional aspects of the devices described herein enhance the abilityto obtain quantitative results. For example, the reservoir designsdiscussed herein above can provide a fixed volume of sample. The fixedvolume permits uniform sampling, which aids in reliable quantitation ofanalyte. Without a fixed volume of sample, quantitation can be, at best,semi-quantitative. In contrast, measurement of a fixed volume permitstrue quantitation.

Further, because the non-porous surface does not retain a dead volume ofsample, essentially all of the applied sample (e.g., essentially all ofa fixed volume of sample) is transferred to the porous carrier materialwherein detection of analyte can occur. Where there is a dead volume ofliquid sample, for example in kits which comprise a porous sampleapplication element or a porous conjugate release element, the amount ofliquid sample retained, and thus not measured for analyte, can vary fromassay to assay, in a manner that does not occur when a reservoir asdescribed herein is employed. It is noted that the space between upperand lower sheets of non-porous material as employed in severalembodiments described herein is also a fixed volume reservoir, i.e., thedevice does not necessarily have to have a reservoir formed by bendingone non-porous surface over on itself as also described.

In addition, the reservoir designs, coupled with the non-porous natureof the zone in which the labeled reagent is temporarily immobilized,also provide a fixed volume for labeled reagent release, resulting in afixed concentration of labeled reagent that does not result in assaysthat lack such a fixed volume reservoir. This design further enhancesthe ability to obtain reliable quantitative results.

The reservoir designs described herein provide a further benefit withregard to quantitative assays in that the filling of the reservoir,e.g., by dipping vertically into a liquid, does not necessarily startthe assay. In one aspect, for example, the reservoir can be designedsuch that the surface of the liquid, when the reservoir is full and thedevice is in the vertical position, is separated from the porous carrierstrip and/or from the reversible immobilization zone. In this aspect,only when the device is laid horizontal does the liquid sample migrateacross the reversible immobilization zone and enter the porous carrierstrip. Thus, such a reservoir design can permit closer control of thetiming of the assay when this is critical, because the assay does notbegin until the device is laid horizontal.

It is to be understood that the measurement of analytes in addition tohCG are encompassed by the devices and methods described herein,including, but not limited to other hormones (e.g., luteinizing hormone(LH), follicle stimulating hormone (FSH), thyroid stimulating hormone(TSR), insulin, pancreatic glucagon and its fragment peptides,parathyroid hormone, calcitonin, adrenocorticoid hormones, growthhormone, etc.), proteins (e.g., viral, fungal or bacterial antigens,antibodies, alphafetoprotein, carcinoembryonic protein, enzymes, PSA,etc.) and chemical analytes such as clinical chemistry analytes anddrugs (including, but not limited to drugs of abuse, e.g., cocaine,heroin, other narcotics, and steroids) and drug metabolites, toxins,etc. That is, the devices described herein can be adapted by one ofskill in the art, given specific binding reagents for the particularanalyte, for the identification of such analytes in liquid samples.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. An analytical device for the single-step determination of thepresence of an analyte in a liquid sample comprising a test stripcomprising: a) a first piece of non-porous solid surface comprising, ina reversible immobilization zone, a reversibly immobilized labeledreagent that binds said analyte to form a complex; b) a porous carriercomprising, in a detection zone, an immobilized reagent that binds saidcomplex, wherein said porous carrier is in contact with said non-poroussolid surface; and c) a second piece of non-porous solid surface placedover (a) and (b).
 2. The device of claim 1 wherein, rather than beingreversibly immobilized upon the first piece of non-porous solidmaterial, said labeled reagent is reversibly immobilized in a reversibleimmobilization zone on the surface of said second piece of non-poroussolid material that faces said first piece of non-porous material, sothat the first piece of non-porous solid material is on the bottom andthe labeled reagent is reversibly immobilized on the underside of saidsecond piece.
 3. A method of detecting an analyte, the method comprisinga) contacting a liquid sample to be tested for said analyte with theanalytical device of claim 1; and b) permitting said liquid sample totraverse said test strip such that said reversibly immobilized labeledreagent is mobilized by the sample and flows to said detection zone,wherein, if analyte is present in said liquid sample, a detectablesignal is generated indicative of the presence of said analyte.
 4. Adevice for the single step quantitative measurement of an analyte in aliquid sample, the device comprising: a) a piece of non-porous solidsurface comprising, in an immobilization zone, a reversibly immobilizedlabeled reagent that binds said analyte to form a complex; and b) aporous carrier comprising a binding reagent and being in contact withsaid non-porous solid surface, wherein said binding reagent is appliedto and immobilized in a detection zone having a longitudinal axisparallel to a flow of a liquid sample, wherein when said liquid samplecomprises the analyte, the binding results in a signal, and wherein thedistance from the point at which said liquid sample entered saiddetection zone to the furthest point in said detection zone at whichsaid signal is detected above background is indicative of the amount ofanalyte present in said sample.
 5. The device of claim 4 wherein saiddetection area is at least 1.5 times as long as it is wide.
 6. Thedevice of claim 4 wherein said detection area extends over at least 50%of the length of said porous carrier.
 7. The device of claim 4 whereinsaid detection area extends over the full length of said porous carrier.8. A method of detecting an analyte, the method comprising a) contactinga liquid sample to be tested for said analyte with the analytical deviceof claim 4; and b) permitting said liquid sample to traverse said teststrip such that said reversibly immobilized labeled reagent is mobilizedby the sample and flows to said detection zone, wherein, if analyte ispresent in said liquid sample, a detectable signal is generatedindicative of the presence or quantity of said analyte.
 9. An analyticaldevice for the single-step determination of the presence or absence ofan analyte in a liquid sample comprising a test strip comprising: anon-porous solid surface comprising, in a reversible immobilizationzone, a reversibly immobilized labeled reagent, that competes with saidanalyte for binding sites present in the detection zone, and a porouscarrier comprising, in a detection zone, an immobilized reagent thatbinds said labeled reagent and analyte in a liquid sample, wherein theabsence of analyte generates a detectable signal, wherein said porouscarrier is in contact with said non-porous surface.
 10. A method ofdetecting an analyte, the method comprising a) contacting a liquidsample to be tested for said analyte with the analytical device of claim9; and b) permitting said liquid sample to traverse said test strip suchthat said reversibly immobilized labeled reagent is mobilized by thesample and flows to said detection zone, wherein, analyte present insaid liquid sample will compete for binding sites in the detection zonewith said labeled reagent, and a detectable signal is generatedindicative of the absence of said analyte.